PROJECT SUMMARY Optogenetic technologies, designed for control of defined elements of neural circuitry with high temporal precision, have come into broad use, including for questions relating to psychiatric disease (e.g. anxiety-related behaviors in freely-moving mammals). Key features of this approach include use of light-activated regulators of transmembrane ion conductance such as channelrhodopsins (ChRs) which are temporally precise as well as straightforward to deliver (for example by viral vectors) to targeted brain regions. We are continuing to expand and improve the optogenetic toolbox through the parent award in order to understand the structure of inhibitory channelrhodopsins that conduct chloride, to use this and other structural data to design red-activated channels for excitation and inhibition, and to leverage these and other powerful optogenetic tools in order to develop and apply all-optical fast closed-loop circuit clamping in behaving mammals. Being able to precisely express molecular tools, including optogenetic ones, in targeted populations of neurons salient to psychiatric disease is critical to extending the utility of these multiply-engineered, next-generation optogenetic tools and cutting-edge, all-optical physiology. This project enables the precise targeting of optogenetic and other molecular tools to highly defined neuron sub-populations and expands a critical approach widely applicable in neuroscience. Lief Fenno, M.D., Ph.D., previously invented a technique, called INTRSECT, that allows for the expression of molecular neuroscience tools in neuron sub-populations defined by multiple parameters (for instance, only neurons expressing both SST AND PV, but not neurons expressing either SST or PV alone). Using protected research time funded by this supplemental program, he will conduct focused research to 1) engineer and validate an expanded toolbox of INTRSECT constructs to include excitatory and inhibitory optogenetic tools, fluorophores, and genetically-encoded calcium indicators; 2) Improve the efficacy of Flp recombinase through iterative engineering and screening of the Flp recombinase recognition cassette; 3) Apply a novel, inexpensive, optical device to acquire long-sought and highly novel data describing the expression kinetics of Adeno- Associated Viruses in vivo; and 4) Extend and refine the targeting resolution of the INTRSECT approach through the novel adaptation of an orthologous recombinase, VCre and apply these new viruses to express optogenetic reagents in neurons based on three separate criteria. This project will produce an improved, expanded, validated, and extended suite of reagents to enable an unlimited number of currently unapproachable projects in neuroscience research.